James B. Hoying - US grants

Specificity in Endothelial Cell Calcium Signaling. The endothelial receives and integrates a variety of stimuli related to blood flow control (i.e. shear stress and vasoactive mediators), inflammation, and growth/repair. Inappropriate or defective endothelial cell responses to these stimuli can lead to a variety of vascular pathologies. Transduction of signals from all of these stimuli relies, in part, on mobilization of intracellular calcium. In large vessel endothelial cells, intracellular calcium pools are managed by two sarco (endothelial cell) plasmic calcium ATPases (SERCA); the uniquely expressed SERCA3 and the ubiquitous SERCA2b pumps. Mice lacking SERCA3 (SERCA3 knockout) are born viable with intact vasculature. However, aortas from these mice fail to respond to the vasodilator acetylcholine. Furthermore, SERCA3-deficient aortic endothelial cells do not generate a calcium transient in response to acetylcholine. The defects in endothelial cell activity present in the SERCA3 knockout mouse occur even though SERCA2b is present. These observations have lead to the hypothesis that endothelial cells utilize distinct intracellular calcium pools, as defined by these two SERCA pumps, to mediate responses to different types of stimuli. The goal of this project is to test this hypothesis, through completion of four specific aims, by demonstrating that the calcium pool maintained by SERCA3 participates in a subset of endothelial cell responses and that this calcium pool is segregated from the SERCA2b-managed pool within the endothelial cell. Specific aims 1 and 2 will characterize the distribution and the spatial organization of the SERCA3- and SERCA2b- managed calcium in the generation of calcium transients and endothelial cell responses induced by four classes of vascular stimuli (shear stress, vasoactivity, inflammation, and vascular repair) in endothelial cells from these mice will provide the experimental basis for these studies. Selective differences observed between cells and vessels of the two mice can be attributed to the absence of calcium stores established by SERCA3 and indicate those processes relying on this calcium pool. Completion of these aims will provide further insight into how specificity in calcium-mediated endothelial cell signal transduction is established and thus provide possible avenues for specific therapies directed at treating vascular disease.

DESCRIPTION (provided by applicant) Regulation of vascular form and function is a dynamic process that depends on the complex integration of responses and activities by vascular cells to a variety of stimuli. In response to these stimuli, the vascular system can mediate numerous physiological processes, rapidly alter vessel diameter to control blood flow, and grow new vessel elements (angiogenesis). Although seemingly separate activities, all of these operations constitute the means by which the vascular system maintains tissue homeostasis and thus represent a single, albeit highly integrative, function. My research career goal is to establish an interdisciplinary vascular research program aimed at understanding the molecular determinants of vascular form and function related to supporting tissue homeostasis. The research development plan to attain this goal is centered around the integration of focused research projects examining specificity in endothelial cell signal transduction, the establishment of vascular tone during vessel remodeling, and the genetic control of vascular growth. The objectives of this plan are to create a highly interactive research team, expand collaborations with accomplished imaging scientists, and gain further expertise in molecular genetics. In large vessel endothelial cells, intracellular calcium pools are managed by two sarco(endothelial cell)plasmic calcium ATPases (SERCA); the uniquely expressed SERCA3 and the ubiquitous SERCA2b pumps. The goal of the research that will examine endothelial cell signal transduction and provide the basis for expanding my collaborative efforts is to test the hypothesis that endothelial cells utilize distinct intracellular calcium pools, as defined by these two SERCA pumps, to mediate responses to different types of stimuli by demonstrating, through completion of four specific aims, that the calcium pool maintained by SERCA3 participates in a subset of endothelial cell and vascular physiological responses. Specific Aims I and 2 will characterize the functional specificity of the SERCA3- and SERCA2b-managed calcium pools within the endothelial cell. Specific Aims 3 and 4 will extend this analysis into the intact animal by examining the role of SERCA3 in vascular function. Mice deficient in the SERCA3 pump and endothelial cells from these mice will provide the experimental basis for these studies. Selective differences observed between cells and vessels of the two mice can be attributed to the absence of calcium stores established by SERCA3 and indicate those processes relying on this calcium pool. Completion of these aims will provide further insight into how specificity, and thus regulation, in signal transduction in endothelial cells is ,established. (End of Abstract)

This proposal focuses on the use of microtubules (MT) as templates for fabricating nanoscale interconnects, interconnect arrays, and networks. MTs are self-assembling, dynamic, and tubular shaped biomolecules with nanometer size diameters and large aspect ratios, made from polymerized tubulin proteins. Their ends are polarized in that each one exhibits unique and specific biochemical moieties. The objectives of the proposed work include establishing the scientific and technical basis for making nano-interconnects from MTs by developing end-specific capping agents to attach to the ends of the MTs, via creation of a combinatorial/phage library. Also, specific ligands to be attached to functionalized metal pads will be identified and synthesized. Attachment control and selectivity will be achieved by creating complementary molecular patterns on the ends of the ligands and capping agents using affinity group - bifunctional reagent complexes and recombinant peptide stretches. Metallization research will provide key insights into the little known area of metal biomolecular interactions with the goal of depositing thin Cu and Au coatings on the interior and/or exterior surfaces of the MTs to improve conductivity. Multiscale modeling and computer simulations will be used to guide research on molecular recognition at the ligand/cap interface and on the effects of geometric and chemical factors on the controlled assembly and disassembly of MT nano-interconnects. The objective of the educational component of the proposed NIRT is to promote the rapid insertion of individuals from under-represented groups into nanotechnology businesses. This objective will be met through the NanoTechnology Track (NTT), a course of study that integrates the scientific, engineering, and business aspects of nanotechnology. This integration emphasizes the transition between research idea and consumer product through a focus on entrepreneurship as taught by the University of Arizona's nationally renowned Berger Entrepreneurship Program. The objectives of the NIRT will be met by completion of a series of planned tasks with measurable outcomes distributed amongst the members of a highly interactive, interdisciplinary team of investigators. This team includes: J. Hoying (cell biology and biomolecular arrays, Biomedical Engineering (BME)), I. Jongewaard (phage display, peptide chemistry, Arizona Health Science Center (AHSC)), R. Guzman (polymer/biomolecule interactions, Chemical and Environmental Engr.), S. Raghavan (surface chemistry, electrochemistry, Materials Science and Engr. (MSE)), B. Zelinski (sol/gel chemistry, MSE), O. Palusinski (microelectronics, packaging and interconnections, Electrical and Computer Engineering (ECE)), P. Deymier (modeling and simulation, MSE) and L. Adamowicz (quantum and computational chemistry, Chemistry). This team will create a virtual education and research unit without traditional departmental boundaries, whose focus is to train diverse undergraduate and graduate students in the scientific and technical multi-linguism needed in today's rapidly evolving field of nanotechnology.

The broader impacts resulting from the proposed activity: The broader impacts of this program revolve around moving biomolecules into the engineering arena. Natural and engineered biomolecules, including proteins, possess properties that add a new dimension to the structure/ processing/ manufacturing/ utilization paradigm, giving them a fabulous long-term potential in a vast range of engineering applications. Through this paradigm, the proposed activity will help enable the electronics industry to push feature sizes down to the nanoscale. Also, this program will establish a new educational initiative, the nanotechnology track (NTT) that will serve as a model for integrating the scientific, engineering, societal and business aspects of nanotechnology into economically sound enterprises.

[unreadable] DESCRIPTION (provided by applicant): Fabricated Microvascular Networks. The importance of an effective vascular supply for tissue health is universally accepted. In developing strategies to build vasculatures for tissue engineering and other therapeutic applications, it is important to recognize that, foremost, the new vasculature must quickly provide sufficient blood flow to the target tissue to preserve cell viability. We have found that new microvessels formed in vitro can begin to carry blood within the first days following implantation. However, flow patterns are atypical and likely ineffective at establishing normoxia until many days later. The delay is primarily due to a lack of organization within the network at the time of implantation and the time needed to develop new mature inflow and outflow pathways. We hypothesize that pre-determining an appropriate network organization prior to implantation would reduce the amount of time needed for the new microvasculature to effectively perfuse a tissue. We have established generic technologies utilizing a direct-write tissue printing tool for patterning and organizing tissue components for tissue engineering applications. We propose to implement this technology to design and fabricate pre-patterned, 3-dimensional microvascular networks with pre-existing inflow and outflow pathways. Also, we will use an in vitro, intravascular-perfusion bioreactor system to establish flow through the networks to further organize and mature the microvascular networks prior to implantation. Computational modeling and physiological analyses serve to direct design strategies and characterize the architectures and functionality of the fabricated vasculatures both in vitro and in vivo. In addition to providing an enabling technology platform for assembling pre-determined microvascular networks, this work will provide a foundation from which to explore the importance of network architectures in vascular function. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Human Microvessel Culture System (hMCS) Angiogenesis-targeting therapies, either inhibiting or promoting new vessel growth, have the potential for treating numerous diseases including cancer, eye diseases, cardiac and peripheral vascular disease, skin disorders, fibro-proliferative diseases, and inflammatory conditions such as arthritis. Furthermore, growing evidence indicates that for many of these diseases combinations of agents targeting multiple different vascular activities are proving more efficacious. As demand and complexity increases for these therapies, there is a growing need for more informative assays of relevant vascular growth and stability. Ideally, such an assay should recapitulate as much of the in vivo biological process as possible, while maintaining experimental simplicity and cost effectiveness. Angiomics, Inc. was founded on proven technology based on the manipulation and culturing of isolated microvessels from a variety of tissues. Utilizing rodent sources, this microvessel culture system has been used extensively and effectively as a more comprehensive experimental and discovery platform for a variety of vascular-related activities and therapies. Because the system utilizes intact microvessels, angiogenesis is more accurately recapitulated in both the culture and in vivo settings. In this system, true neovessels sprout and grow from intact microvessels within a 3-D matrix environment. Consequently, all angiogenesis-relevant activities/elements including perivascular cell behavior are present and integrated in the assay. The overall objective of this proposal is to adapt this proven and versatile microvessel culture system to support human microvessel viability and activity to add utility and value to the platform. It is guided by two Objectives tha 1) identify media components, guided by the rodent system, and 2) evaluate combinations of candidate growth factors or hypoxia factors that will support human microvessel viability and angiogenesis in culture.

SUMMARY Neovessel guidance in angiogenesis Vascular connectivity between adjacent vessel beds within and between tissue compartments is an essential aspect of any successful neovascularization process. To establish new connections, growing neovessels must locate other vascular elements during angiogenesis, often crossing matrix and other tissue-associated boundaries and interfaces. This is perhaps best highlighted in situations of tissue grafting (e.g. tissue free flaps) during which the vasculature of the graft must cross the graft-host tissue interface before connecting to the surrounding host circulation in order for the graft to survive. An inability for a tissue graft vasculature to connect to the host vasculature is the primary cause for tissue graft failure and necrosis. How growing neovessels traverse any tissue interface, whether part of the native tissue structure or secondary to a grafting procedure, is not known. In a series of preliminary experiments, we have determined that actively growing neovessels are unable to spontaneously cross a stroma-stroma interface during angiogenesis. Our published findings that tissue stromal biomechanics, specifically the biophysical aspects of the matrix, has a profound influence on the direction and branching of growing neovessels suggests that this interface is biomechanically incompatible with interface invasion. In addition, we have evidence that a sub-population of tissue-resident, stromal macrophages facilitates neovessel invasion of the stromal interface during angiogenesis through a VEGF-A-dependent process. Importantly, it appears that stromal cells need to migrate across the interface in order to promote interface invasion by the neovessels, suggesting that spatial gradients of VEGF-A are required. Based on these observations, we hypothesize that the graded angiogenic factor signals overcome the biomechanical barriers to directed angiogenesis caused by a stroma-stroma interface to promote interface neovascular invasion. The project will involve a combination of in vitro and computational models of angiogenesis across tissue-tissue boundaries to test this hypothesis and determine the mechanism by which stromal cells use VEGF-A signaling to regulate neovessel behavior in coordination with stroma biomechanical dynamics. These studies will provide new insights into a poorly understood aspect of vascular biology and tissue-vascular dynamics as well as create opportunities for therapeutic strategies to facilitate tissue healing, improving angiogenesis-based treatments, and tissue grafting/transplantation.